Fermentation mammalian cell cultures

Insect cell systems represent multiple advantages compared with mammalian cell cultures (1) they are easier to handle (Table 2.1) (2) cultivation media are usually cheaper (3) they need only minimum safety precautions, as baculovirus is harmless for humans (4) they provide most higher eukaryotic posttranslational modifications and heterologous eukaryotic proteins are usually obtained in their native conformation (5) the baculovirus system is easily scalable to the bioreactor scale. However, because of the viral nature of the system, continuous fermentation for transient expression is not possible - the cells finally die. 

In spite of several drawbacks (i.e. expensive and laborious handling procedures, low space-time yields (Table 2.1), high demand on biosafety, potential contaminations, limited applicability for continuous fermentations [129], and problems obtaining the same glycosyla-tion profile from batch to batch [130]), mammalian cell cultures are widely used for small-scale expression and more recently even on a multi-cubic-meter scale. The system works like insect... 

Probably the most exciting aspect of molecular farming from the industrial perspective is the low initial capital investment compared with mammalian cell culture production. The reduced capital input reflects the lower costs of laboratory equipment and materials for plant molecular biology. For example, the costs involved in establishing small, non-sterile greenhouse facilities are dwarfed by those required for a sealed pilot fermentation plant. 

Mammalian cell culture is more technically complex and more expensive than microbial cell fermentation. Therefore, it is usually only used in the manufacture of therapeutic proteins that show extensive and essential post-translational modifications. In practice, this usually refers to glycosylation, and the use of animal cell culture would be appropriate where the carbohydrate content and pattern are essential to the protein s biological activity, its stability or serum half-life. Therapeutic proteins falling into this category include EPO (Chapter 10), the gonadotrophins (Chapter 11), some cytokines (Chapters 8-10) and intact monoclonal antibodies (Chapter 13). 

As described in Section 4.2, traditional biotech processes—namely, biocatalysis and microbial fermentation—are used for the production of small molecules, whereas the modern cell culture methodology allows the production of HMW biopharmaceuticals. A growth rate of 10-15% per annum is expected for the biotechnological contribution, while the average increase of the pharmaceutical market remains below 10%. In terms of technologies, the demanding mammalian cell cultures are expected to grow fastest, followed by microbial fermentation. 

Transgenic plant systems have the potential to produce recombinant proteins on a commodity scale (Kusnadi et al., 1997) due to the low cost of growing plants and because scale-up of production simply requires sewing seeds over a greater field area. As such they offer almost unlimited scalability (Giddings, 2001). It is estimated by Kusnadi et al. (1997) that transgenic plants can produce pharmaceutical proteins at between 10 and 50-fold lower cost than microbial fermentation systems, and 1,000 times lower than mammalian cell culture systems (Hood et al., 2002). 

Biopharmaceuticals that are industrially produced by microbial (bacterial or yeast) fermentation include insulin, human growth hormone, hepatitis B surface antigen vaccine, alpha interferon, beta interferon, gamma interferon, granulocyte colony stimulating factor, and interleukin-2. Table 30.8 lists some of the major biopharma products, some of which are produced by mammalian cell culture. 

Zhu, M.M., E.S. Lee, W.R. Hermans, and D.J. Wasilko. 2001. Overview and serum-free medium development for mammalian cell culture. Fourth Conference on Recent Advances in Fermentation Technology (RAFTIV), Nov 11-13, Long Beach, CA. 

The ionophores and several other specialty products are included in Table 13.8 for comparison purposes. Products of mammalian cell culture such as plasminogen activator and erythropoietin are included as fermentation products in this listing because they are normally manufactured by cellular processes in bioreactors. Aside from the five commodity chemicals in this table, the most dramatic change in the commercial chemicals produced by fermentation results from the impact of genetic engineering and recombinant DNA methods on the specialty products. Antibiotics and biopolymers (hormones, enzymes, etc.) with molecular structures too complex for conventional chemical synthesis will continue to be manufactured by microbial processes (Hinman, 1993). 

Werner RG Noe W (1993b) Mammalian cell cultures. Part II Genetic engineering, protein glycosylation, fermentation and process control. Arzneimittel Forschung 43 1242-1249. 

For most aerobic fermentation processes, maintenance of absolute sterility is critical to cell growth and biomass production. Culture growth rate determines susceptibility to culture contamination. Mammalian cells divide in a day, while microbials divide in an hour or less. The difference in growth rate makes slower-growing mammalian cell cultures more susceptible... 

For each parameter, the pH, DO (dissolved oxygen), ORP (oxidation-reduction potential), temperature, agitation speed, culture volume and pressure can be measured with sensors located in the fermenter. The output of the individual sensors is accepted by the computer for the on-line, continuous and real-time data analysis. Information stored in the computer control system then regulates the gas flow valves and the motors to the feed pumps. A model of a computer control system is shown in Fig. 11. The computer control systems, like the batch systems for mammalian cell culture, seem to level out at a maximum cell density of 10 cells/ml. It may be impossible for the batch culture method to solve the several limiting factors (Table 10) that set into high density culture where the levels are less than 10 cells/ml. 

The metabolites glutamine and glucose are the main substrates of mammalian cell culture. Nevertheless, even if they are not limited in oxygen, proliferating mammalian cells do not completely oxidize their main substrates to CO2 but produce the fermentative product lactate. This observation, termed Warburg effect, is hypothesized to be a result of the needs of proliferating cells for precursor metabolites instead of maximum ATP formation via complete... 

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